Histone deacetylase 1 (HDAC1) (IC50 = 3.3 nM for immunoprecipitated HDAC1 complexes)
Histone deacetylase 8 (HDAC8) (IC50 = 23 nM for recombinant HDAC8)
Class I HDACs in rat liver extract (IC50 = 86 nM for unselective MAL substrate; IC50 = 51 nM for HDAC1-selective B61 substrate)
Class IIb HDAC6 in rat liver extract (IC50 = 541 nM for HDAC6-selective B12 substrate)[1]

JNJ-16241199's effect on leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CLL), chronic myeloid leukemia (CML), acute, and myeloma cell proliferation (IC50 = 15). JNJ-16241199 inhibits primary human mammary epithelial cells' proliferation with an IC50 value of 32 nM, while being insensitive to HMEC cells that are at rest and not actively proliferating (IC50 value of 7815 nM) [1]. -486 nanometers) [1]. In A2780 cells, -16241199 (0.1, 0.3, 1 μM, 24-48 h) can both stimulate and inhibit angiogenesis [1].

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R306465 inhibited the proliferation of A2780 ovarian carcinoma cells with an IC50 of 39 nM, measured by an MTT assay after 4 days of incubation.[1]
R306465 demonstrated broad-spectrum antiproliferative activity against a panel of solid tumor cell lines (ovarian, lung, colon, breast, prostate) with IC50 values ranging from 30 to 300 nM, as measured by MTT or Alamar Blue assays.[1]
R306465 also potently inhibited the proliferation of various haematological malignancy cell lines (acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphoblastic leukaemia, chronic myeloid leukaemia, lymphoma, myeloma) with IC50 values ranging from 15 to 486 nM.[1]
R306465 inhibited the growth of primary human umbilical vein endothelial cells (HUVECs) with an IC50 of 186 nM, as measured by a BrdU incorporation assay.[1]
In A2780 cells, R306465 induced histone H3 acetylation and upregulated p21waf1/cip1 protein expression at concentrations as low as 100 nM, as detected by western blot analysis after 24 hours of incubation. Tubulin acetylation and Hsp70 induction (markers of HDAC6 inhibition) were observed only at higher concentrations (1 µM), indicating selectivity for class I HDACs over HDAC6.[1]
R306465 induced cell cycle arrest (decrease in S phase, increase in G1 phase) and apoptosis (increase in sub-G1 fraction and Annexin V-positive cells) in A2780 ovarian carcinoma cells in a concentration-dependent manner after 24-48 hours of treatment, as analyzed by flow cytometry.[1]
R306465 induced DNA fragmentation in A2780 cells at 100 nM, as assessed by TUNEL staining.[1]
R306465 inhibited angiogenesis in the rat aortic ring assay, significantly reducing microvessel area by 67% at 300 nM compared to controls after 8 days of culture.[1]
Combination studies showed that R306465 exhibited additive or synergistic effects with Bortezomib in a broad panel of haematological tumor cell lines (e.g., synergy in MOLT-4, SUP-B15, THP-1, K-562, U-937, Namalwa, Raji, RPMI 8226 cells), as calculated by combination index (CI) values.[1]

In orthotopic xenograft tumor models of A2780 ovarian cancer, H460 lung cancer, and HCT116 cancer, JNJ-16241199 (10–40 mpk daily for 28 days, po) suppresses the growth of tumors [1].

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Oral administration of R306465 once daily at well-tolerated doses (10, 20, 40 mg/kg) for 28-32 days significantly inhibited the growth of A2780 ovarian, H460 lung, and HCT116 colon carcinoma xenografts in immunodeficient mice. Maximal tumor volume reductions of 76-87% were observed at 20 and 40 mg/kg doses in the A2780 model.[1]
A single oral dose of R306465 (40 mg/kg) induced histone H3 acetylation in A2780 ovarian xenograft tumor tissue, as detected by immunofluorescence 4 hours post-dose. Maximal and homogeneous H3 acetylation throughout the tumor was observed after 2 days of treatment.[1]
In A2780 tumors from mice stably transfected with a p21waf1/cip1 promoter-driven ZsGreen reporter, a single oral dose of R306465 (40 mg/kg) activated the p21 promoter, as evidenced by increased ZsGreen fluorescence in tumor sections 24 hours post-dose. The responding cell clusters were distributed evenly throughout the tumor and were not localized with vasculature.[1]

For HDAC1 activity, HDAC1 was immunoprecipitated from A2780 ovarian tumor cell lysates using anti-HDAC1 antibody conjugated to protein A agarose beads. The immunoprecipitated complexes were incubated with a [³H]-acetyl-labeled histone H4 peptide fragment as substrate in assay buffer. Test compounds were pre-incubated with the enzyme for 10 minutes at room temperature before substrate addition. The reaction proceeded at 37°C for 45 minutes, was quenched with stop buffer, and the released [³H]acetic acid was extracted with ethyl acetate and quantified by scintillation counting.[1]
For recombinant HDAC8 activity, a commercial colorimetric/fluorimetric activity assay kit was used according to the manufacturer's instructions.[1]
For HDAC isotype selectivity in rat liver extract, partially purified extracts were incubated with different fluorogenic substrates: an unselective substrate (MAL), an HDAC1-selective substrate (B61), and an HDAC6-selective substrate (B12). Deacetylation rates were measured fluorometrically.[1]

Cell cycle analysis [1]
Cell Types: human ovarian cancer cell A2780
Tested Concentrations: 0, 0.1, 0.3, 1 μM
Incubation Duration: 24 hrs (hours) or 48 hrs (hours)
Experimental Results: At 300 nM, the S phase diminished and the G1 phase increased in parallel, but after 24 hrs (hours) The sub-G1 fraction of cells increased at 1 μM. After 48 hrs (hours), there was an increase in the sub-G1 phase at all active concentrations starting from 100 nM.

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For cell proliferation assays (MTT), cells were seeded in 96-well plates and allowed to adhere. After 24 hours, compounds diluted in culture medium (final DMSO ≤ 0.1%) were added and incubated for 4 days. MTT reagent was added, formazan crystals were solubilized, and absorbance was measured. IC50 values were calculated by non-linear regression.[1]
For cell cycle analysis, A2780 cells were treated with compound, collected, fixed, stained with propidium iodide, and analyzed by flow cytometry. Cell cycle distribution was determined using analysis software.[1]
For apoptosis assessment (Annexin V/PI staining), treated A2780 cells were collected, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry.[1]
For TUNEL staining, treated A2780 cells were fixed, permeabilized, and incubated with a solution containing fluorescein-dUTP and terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks. Incorporated fluorescence was detected.[1]
For western blot analysis, A2780 cells were treated, lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against acetylated H3, total H3, p21, acetylated tubulin, total tubulin, Hsp70, and c-raf. Detection was performed using chemiluminescence or fluorescence.[1]
For the rat aortic ring angiogenesis assay, thoracic aortic rings from rats were embedded in fibrin gels in serum-free media. Compounds were added at culture initiation and refreshed periodically. After 8 days, microvessel outgrowth was quantified using automated image analysis.[1]
For HUVEC proliferation (BrdU assay), cells were seeded in low-serum medium, stimulated with growth supplement, and treated with compounds. BrdU was added, and its incorporation over 24 hours was measured using a commercial kit.[1]

Animal/Disease Models: Human A2780, H460 and HCT116 orthotopic xenograft tumor model [1]
Doses: 10-40 mpk/day for 28 days
Route of Administration: po (oral gavage)
Experimental Results: Induction of H3 in A2780 ovarian tumor tissue Acetylation and p21waf1, cip1 promoter activity. Tumor volume reduction in three orthotopic xenograft tumor models. In the human A2780 orthotopic xenograft tumor model, the final tumor volume was diminished to a maximum of 76-87%.

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For antitumor efficacy studies, immunodeficient nude mice were inoculated subcutaneously with human tumor cells (A2780 ovarian, H460 lung, or HCT116 colon carcinoma; 10⁷ cells/mouse). Starting from day 4 post-inoculation, mice were treated orally once daily (QD) with R306465 (formulated at 2 mg/mL in 20% hydroxypropyl-β-cyclodextrin, pH 8.7) at doses of 10, 20, or 40 mg/kg for 28 (A2780) or 32 (H460, HCT116) days. Tumor dimensions were measured regularly, and volume was calculated.[1]
For pharmacodynamic studies (H3 acetylation and p21 promoter activation), mice bearing A2780 xenografts (or A2780-p21-ZsGreen reporter xenografts) were given a single oral dose of R306465 (40 mg/kg) or vehicle. Tumors were harvested at specified time points (e.g., 4h, 24h) post-dose for immunofluorescence or fluorescence microscopy analysis.[1]

[1]. R306465 is a novel potent inhibitor of class I histone deacetylases with broad-spectrum antitumoral activity against solid and haematological malignancies. Br J Cancer. 2007;97(10):1344-1353.

R306465 has been used in clinical trials for cancer treatment research.

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R306465 (also known as JNJ-16241199) is a novel hydroxamic acid histone deacetylase (HDAC) inhibitor. It is selective for class I HDACs (especially HDAC1 and HDAC8) but non-selective for class IIb enzyme HDAC6, which distinguishes it from other clinical HDAC inhibitors such as vorinostat (SAHA, which preferentially inhibits HDAC6) and pabisostat (broad-spectrum). Its antitumor mechanism involves the inhibition of class I HDACs, resulting in histone hyperacetylation, altered gene expression (e.g., p21 upregulation), cell cycle arrest, apoptosis, and anti-angiogenic effects. In preclinical models, it has shown broad-spectrum activity against solid tumors and hematologic malignancies and is administered orally. [1]

C19H19N5O4S

413.45026

413.115

604769-01-9

10309899

White to off-white solid powder

1.5±0.1 g/cm3

1.689

2.06

2

8

4

29

669

0

O=C(C1=CN=C(N2CCN(S(=O)(C3=CC=C4C=CC=CC4=C3)=O)CC2)N=C1)NO

MUTBJZVSRNUIHA-UHFFFAOYSA-N

InChI=1S/C19H19N5O4S/c25-18(22-26)16-12-20-19(21-13-16)23-7-9-24(10-8-23)29(27,28)17-6-5-14-3-1-2-4-15(14)11-17/h1-6,11-13,26H,7-10H2,(H,22,25)

N-hydroxy-2-(4-(naphthalen-2-ylsulfonyl)piperazin-1-yl)pyrimidine-5-carboxamide

R 306465 JNJ-16241199R306465 JNJ16241199 R-306465JNJ 16241199.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)